Latent TGFß-binding proteins regulate UCP1 expression and function via TGFß2.

OBJECTIVE: Activation of brown adipose tissue (BAT) in humans has been proposed as a new treatment approach for combating obesity and its associated diseases, as BAT participates in the regulation of energy homeostasis as well as glucose and lipid metabolism. Genetic contributors driving brown adipogenesis in humans have not been fully understood. METHODS: Profiling the gene expression of progenitor cells from subcutaneous and deep neck adipose tissue, we discovered new secreted factors with potential regulatory roles in white and brown adipogenesis. Among these, members of the latent transforming growth factor beta-binding protein (LTBP) family were highly expressed in brown compared to white adipocyte progenitor cells, suggesting that these proteins are capable of promoting brown adipogenesis. To investigate this potential, we used CRISPR/Cas9 to generate LTBP-deficient human preadipocytes. RESULTS: We demonstrate that LTBP2 and LTBP3 deficiency does not affect adipogenic differentiation, but diminishes UCP1 expression and function in the obtained mature adipocytes. We further show that these effects are dependent on TGFß2 but not TGFß1 signaling: TGFß2 deficiency decreases adipocyte UCP1 expression, whereas TGFß2 treatment increases it. The activity of the LTBP3-TGFß2 axis that we delineate herein also significantly correlates with UCP1 expression in human white adipose tissue (WAT), suggesting an important role in regulating WAT browning as well. CONCLUSIONS: These results provide evidence that LTBP3, via TGFß2, plays an important role in promoting brown adipogenesis by modulating UCP1 expression and mitochondrial oxygen consumption.

Anti-inflammatory properties of bone morphogenetic protein 4 in human adipocytes.

BACKGROUND: Obesity is characterized by increased adipocyte number and size as well as white adipose tissue (WAT) inflammation, which is fundamental for the development of insulin resistance and type-2 diabetes. These processes, regulated by various endocrine, paracrine and autocrine factors, are extensively studied with the hope to interfere and to inhibit weight gain and related complications in obese patients. Recent data suggest an important role of bone morphogenic protein 4 (BMP4) in the regulation of adipogenesis and development of obesity. BMP4 is a growth factor of the transforming growth factor-ß superfamily. Initially, BMPs were identified as inducers of ectopic bone formation. It is now apparent, however, that these proteins have different pleiotropic developmental actions and including playing a role in white adipogenesis. METHODS AND RESULTS: Here, we demonstrate that the expression of BMP4 in human WAT is negatively correlated to body mass index and to the expression of pro-inflammatory cytokines. In vitro, BMP4 expression in cultured human adipocytes is upregulated after induction of differentiation. Cells treated with exogenous BMP4 increased peroxisome proliferator-activated receptor γ (PPARγ) expression and significantly reduced the expression of pro-inflammatory cytokines including tumor necrosis factor α (TNF-α) and monocyte chemoattractant protein 1. TNF-α treatment of fully differentiated adipocytes resulted in downregulation of the expression of adipogenic genes and elevated expression of pro-inflammatory cytokines. Exogenous BMP4 addition significantly reduced the negative effect of TNF-α on the expression profile of adipocytes. Finally, treatment of human adipocytes with exogenous BMP4 reduced the adipocytes' chemoattractant potential and the migration of monocytes toward adipocyte-conditioned medium. CONCLUSIONS: These results indicate that BMP4 is an important anti-inflammatory molecule, which may act through PPARγ and reduces TNF-α-mediated pro-inflammatory cytokine production in human adipocytes. Through its anti-inflammatory potential, BMP4 may serve as a protective factor for inflammation-related diseases such as insulin-tolerance or type-2 diabetes.

Weighing the options in the pharmacotherapy of obesity.

In industrial nations the most common form of malnutrition is obesity. Obesity is a chronic disease associated with increased morbidity and mortality. Unfortunately, the simplest treatment for obesity, increasing physical activity and reducing caloric intake, for the vast majority of patients is completely ineffective over the long term. Weight lost is quickly regained, and body weights after intervention often exceed pretreatment levels. Early pharmacotherapy had been successful in reduction and maintenance of weight loss; unfortunately, unforeseen side effects contributed to an unacceptable risk/benefit balance that necessitated the withdrawal of many of these agents. Current pharmacotherapy is limited, with only 2 compounds accepted for long-term use for the treatment of obesity. Both sibutramine and orlistat contribute to modest weight loss and maintenance and reduce obesity-associated risk factors. Beginning with the discovery of leptin in the mid-1990s, a renaissance in obesity research has taken place. This research has provided more detailed insights into the regulation of energy homeostasis. The central feedback regulation loops involving the leptin and melanocortinergic pathways have provided several targets for therapeutic intervention, including leptin analogs and melanocortin agonists. Several peripheral targets including uncoupling proteins and beta3-adrenergic receptor are also being explored for their anti-obesity potential. Therefore, it can be expected that new pharmacotherapies will be available to help combat the epidemic of obesity.

The role of NPY in metabolic homeostasis: implications for obesity therapy.

Neuropeptide Y (NPY) is a 36 amino acid amidated peptide which has now emerged as an important regulator of feeding behaviour. Upon intracerebroventricular (icv.) administration, NPY produces a pronounced feeding response in a variety of species. The actions of NPY are believed to be mediated by a family of receptor subtypes named Y1 - y6. Recent studies suggest that the Y1 and Y5 receptor subtypes are intimately involved in NPY induced feeding. This review presents preclinical data obtained with receptor subtype selective agonists and antagonists as well as findings from knockout mice. These new data suggest that NPY receptor antagonists may become an additional option for treating human obesity.

Hammerhead ribozymes that selectively cleave the NPY Y1, Y4, and Y5 receptor full-length RNA.

The concept of exploiting the ribozyme catalytic center for cleaving a specific target RNA transcript was applied to the design of selective ribozymes for the rat Y1, Y4, and Y5 receptor subtypes. Ribozymes selective for the neuropeptide Y (NPY) receptor subtypes were designed and chemically modified. Recognition sites were selected both according to the extent of their sequence homology between the receptor subtypes and according to the localization within single-stranded regions accessible for hybridization. Stability of the ribozymes against nucleolytic activities was increased by introducing 2'-O-methylribonucleosides and 3'-terminal modifications, such as inverted ends or dideoxynucleosides. Ribozymes cleaving the full-length rat Y1, Y4 (1200 nt), and Y5 receptor mRNA (2200 nt) were identified. The specificity of the recognition sites and the subtype selectivity of the ribozyme-mediated cleavage was demonstrated.

Magnetic resonance properties of ex vivo breast tissue at 1.5 T.

The magnetic resonance absorption spectrum, T1 and T2 relaxation time distributions, and magnetization transfer properties of ex vivo breast tissue have been characterized at 1.5 T and 37 degrees C. The fraction of fibroglandular tissue within individual tissue samples (n = 31) was inferred from the tissue volumetric water content obtained by integration of resolvable broad-line fat and water resonances. The spectroscopically estimated water content was strongly correlated with that extracted enzymatically (Pearson correlation coefficient 0.98, P < < 0.01), which enabled the assignment of principal relaxation components for fibroglandular tissue (T2=0.04+/-0.01, T1=1.33+/-0.24 s), and for adipose tissue (T2=0.13+/-0.01, T1=0.23+/-0.01 s, and T2=0.38+/-0.03, T1=0.62+/-0.16 s). Th e relaxation components for fibroglandular tissue exhibited strong magnetization transfer, whereas those for adipose tissue showed little magnetization transfer effect. These results ultimately have applicability to the optimization of clinical magnetic resonance imaging and research investigations of the breast.

Comparison of the enzyme activities between the thickest and the thinnest parts of the abdominal pannus.

OBJECTIVE: According to some reports, there are variations in metabolism in adipocytes from different areas of the body. The purpose of this study was to determine if there are differences in some of the lipid assimilating enzyme activities between the thickest (overhang) and the thinnest (upper margin) parts of the abdominal pannus. METHODS: The abdominal panniculectomy activities of sn-glycerol-3-phosphate dehydrogenase (31 subjects, spectrophotometric method), fatty acid synthetase (14 subjects, spectrophotometric method) and lipoprotein lipase (18 subjects, radioactive method) were determined in the thickest and the thinnest parts of the pannus of lipectomy patients. RESULTS: The enzyme activities were as follows: sn-glycerol-3-phosphate dehydrogenase: thickest: 2083 +/- 227.7 nm/mg/min; thinnest: 2084 +/- 208.3 nm/mg/min (p < 0.098, T = 0.02). Fatty acid synthetase: thickest: 22.0 +/- 3.9 microns/mg/min; thinnest 25.9 +/- 6.9 microns/mg/min (p < 0.36, T = 0.94). Lipoprotein lipase: thickest: 0.70 +/- 0.11% of control; thinnest: 0.61 +/- 0.14% of control (p < 0.47, T = 0.75). Thus no differences in specific enzyme activities were found between the two sites studied. CONCLUSIONS: There was no difference in the activity of the enzymes studied at the thickest and the thinnest part of the pannus.

Increasing vimentin expression associated with differentiation of human and rat preadipocytes.

Alterations in the cytoskeletal apparatus constitute some of the earliest changes during assumption of an adipogenic phenotype. We examined three major cytoskeletal elements, beta-actin, alpha-tubulin and vimentin, during adipogenesis in euploid cells from human and rat adipose tissue. As reported with 3T3 sub-lines, mRNA level for beta-actin and alpha-tubulin were decreased upon differentiation. However, in contrast to reports with 3T3 cells, levels of vimentin were increased during differentiation. Furthermore, immunological analyses confirmed that there was no decrease in vimentin protein levels during adipogenic development. As well as highlighting a difference between 3T3 cell lines and preadipocytes isolated from fat depots, these studies indicate that the pattern of cytoskeletal gene expression undergoes complex changes early during preadipocyte differentiation.

Biochemistry of Short-Chain Alkanes (Tissue-Specific Biosynthesis of n-Heptane in Pinus jeffreyi).

Short-chain (C7-C11) alkanes accumulate as the volatile component of oleoresin (pitch) in several pine species native to western North America. To establish the tissue most amenable for use in detailed studies of short-chain alkane biosynthesis, we examined the tissue specificity of alkane accumulation and biosynthesis in Pinus jeffreyi Grev. & Balf. Short-chain alkane accumulation was highly tissue specific in both 2-year-old saplings and mature trees; heart-wood xylem accumulated alkanes up to 7.1 mg g-1 dry weight, whereas needles and other young green tissue contained oleoresin with monoterpenoid, rather than paraffinic, volatiles. These tissue-specific differences in oleoresin composition appear to be a result of tissue-specific rates of alkane and monoterpene biosynthesis; incubation of xylem tissue with [14C]sucrose resulted in accumulation of radiolabel in alkanes but not monoterpenes, whereas incubation of foliar tissue with 14CO2 resulted in the accumulation of radiolabel in monoterpenes but not alkanes. Furthermore, incubation of xylem sections with [14C]acetate resulted in incorporation of radiolabel into alkanes at rates up to 1.7 nmol h-1 g-1 fresh weight, a rate that exceeds most biosynthetic rates reported with other plant systems for the incorporation of this basic precursor into natural products. This suggests that P. jeffreyi may provide a suitable model for elucidating the enzymology and molecular biology of short-chain alkane biosynthesis.

Increased obese mRNA expression in omental fat cells from massively obese humans.

Obesity presents a significant challenge to the general health of affluent nations in terms of the number of people affected, the serious associated maladies and the lack of effective treatments. While common wisdom has held that obesity results from 'gluttony and sloth', a number of studies have indicated physiological causes of underlying the pathogenesis of obesity, with the degree of adiposity having a strong genetic component. Recently, the obese gene in the ob/ob mouse was cloned, along with its human homologue. The specific production of the obese protein by adipose tissue suggested that it may function in a feedback loop from fat tissue to the hypothalamus to control energy intake and/or energy expenditure, and that it may play a role in the pathogenesis of human obesity. In this study we report that obese mRNA expression is elevated in ex vivo omental adipocytes isolated from massively obese humans in the absence of an identifiable mutation. Therefore, we speculate that this increased expression may suggest that the massively obese are insensitive to the putative regulatory function(s) of the obese gene product.

Decreasing expression of a gene encoding a protein related to basic fibroblast growth factor during differentiation of human preadipocytes.

Pursuant to our findings that pituitary basic fibroblast growth factor (bFGF) stimulates the replication of preadipocytes and inhibits their differentiation, we have studied the changes in expression of a bFGF-related mRNA during differentiation. Human omental preadipocytes were grown in primary culture and induced to differentiate with chemically defined serum-free medium. The differentiation process was assessed by monitoring the rise of glycerophosphate dehydrogenase (GPDH) activity, while mRNA expression for a bFGF-related protein(s) and GPDH was examined by amplifying the respective target sequences by polymerase chain reaction. In the case of all cell strains, differentiated preadipocytes revealed much lower expression of bFGF-related mRNA than undifferentiated preadipocytes. Concurrently, the expression of the GPDH mRNA rose significantly. The finding that the expression of the bFGF-related protein is decreased appreciably during adipose differentiation is consonant with its proposed function to expand and maintain adipose cells in a relatively undifferentiated state.

Cellular and molecular factors in adipose tissue growth and obesity.

Heparin-binding growth factors related to basic fibroblast growth factor are major determinants of the cellular clonal composition of adipose tissue. By providing and maintaining varying complements of preadipocytes in different fat depots, these factors contribute to the varying sizes and functions of different regions, including the hypercellularity in appreciable obesity. Thus, differing levels and activities of the heparin-binding growth factors contribute to variations in depots within the same individual and between individuals, in lean and obese states. In contrast to regional differences in adiposity, which are accounted by factors resident in adipose tissue, we believe that obesity results from a generalized energy overload. According to our concept, there are genetic variations in cytoskeletal activity and thus differing quantities of energy are utilized for biomechanical processes. In a reciprocal relationship, the higher the cytoskeletal activity, the lesser the energy available for chemical energy storage, mainly in the form of triglyceride in adipocytes. At the extreme of "supermassive" obesity, a mutation in a gene related to a cytoskeletal protein would lead to appreciable dampening of cytoskeletal activity, with consequently the greatest quantity of energy remaining available for eventual triglyceride storage. Moreover, the new concept, for which we have have increasing experimental evidence, invokes a hypothalamic-efferent neural-cytoskeletal pathway, which would modulate the activity of the cytoskeleton.

Augmented production of heparin-binding mitogenic proteins by preadipocytes from massively obese persons.

Basic fibroblast growth factor (bFGF) stimulates the replication of preadipocytes and inhibits their differentiation. In this study we explored whether the same or related polypeptides were produced locally and acted by paracrine/autocrine mechanisms in adipose tissue. Omental preadipocytes from 7 lean and 10 massively obese (> 170% reference) subjects were grown to confluence in subculture. Total RNA was hybridized with a synthetic deoxynucleotide for human bFGF. In the case of all cell strains, there was expression of two major bFGF transcripts, 7.0 and 3.7 kb. Although there was considerable variation in the degree of expression, preadipocytes from massively obese subjects revealed much greater expression than did cells from the lean (P < 0.001). In studies of conditioned media prepared with preadipocytes, the presence of proteins belonging to the heparin-binding (fibroblast) growth factor family was indicated by Western blot analysis, for a 66-kD protein with anti-(1-24)bFGF, and for a 32-kD protein with anti-(40-63)bFGF antibodies. The relative quantity of the 66-kD protein correlated with body mass index at r = 0.72. bFGF-related proteins probably function normally to maintain an appropriate complement of adipocyte precursors. The augmented expression of heparin-binding growth factors in preadipocytes from some massively obese people probably contributes to the excessive cellularity of their fat depots.

Accumulation of starch in Chlamydomonas reinhardtii flagellar mutants.

Paralyzed flagellar mutants pf-1, pf-2, pf-7, and pf-18 of the green alga Chlamydomonas reinhardtii (Dangeard) were shown to store a significantly greater amount of starch than the motile wild type 137c+. The increase in starch storage was significant relative to protein, chlorophyll, and cell number. Analysis of average cell size revealed that the paralyzed mutants were larger than the wild type. This increase in storage molecule accumulation supports an inverse relationship between chemical energy storage and energy utilization for biomechanical/motile cellular functions. Chlamydomonas reinhardtii provides a useful model for studies of the role of cytoskeletal activity in the energy relationship and balance of organisms.